There is a continuous need in medical practice, research and diagnostic procedures for rapid, accurate, quantitative determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, seminal fluids and other biological fluids has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be detected and receptors reactive with that substance. Radioactive or enzyme labels have been used to detect the resulting reactive complex.
One particular type of test which has been developed is an agglutination test which is useful for the detection of antigens which have multiple sites for antibody reactivity. In such a test, anti-body molecules can be bound in a suitable fashion to water-insoluble particles. Antibody-antigen reaction at multiple sites causes the particles to agglutinate and precipitate. Suitable separation and detection means have been devised to make the agglutinate readily observable, including for example, the use of particles containing a tracer material as described in copending U.S. Ser. No. 98,583, filed Sept. 18, 1987 by Sutton, Littlehale and Danielson, now U.S. Pat. No. 4,997,772 and in references cited therein.
Another useful method for detecting biological substances in fluids is what is known in the art as a "sandwich" assay. Such an assay involves "sandwiching" the compound of interest (such as an antigen) with two or more receptor molecules (such as antibodies) which complex with the compound at different and noninterfering sites. Examples of such assays are described, for example, in U.S. Pat. No. 4,486,530 (issued Dec. 4, 1984 to David et al). In most sandwich assays, one or more of the receptor molecules are suitably immobilized on an insoluble carrier such as small particles, membranes, plates, test wells or similar objects.
Attachment of antibodies or receptor molecules to insoluble carrier materials has been achieved in the past in a number of ways. Early work relied on adsorption of the molecules, but it was realized that adsorption is generally not a strong method of attachment. Later researchers found that the molecules could be covalently attached by reaction of certain functional groups of the molecules with specially designed reactive groups on the carrier material. For example, proteins have been attached by reacting carboxy groups of particles or supports with an activating compound which renders the groups reactive with amino groups of a protein. Carbodiimides are examples of useful activating compounds.
Avidin is a protein found in egg whites. Biotin (hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid), also known as Vitamin H, is a relatively small water-soluble molecule. These materials are known to react specifically with each other to form a very strong and stable complex in which each of the four subunits of avidin binds a biotin molecule. This strong binding is maintained even when either biotin or avidin or both are bound covalently to other materials. The reaction has been used to enhance agglutination of erythrocytes and provided researchers with a means for various biochemical and diagnostic studies.
U.S. Pat. No. 4,298,685 (issued Nov. 3, 1981 to Parikh et al) describes a competitive immunoassay in which antibodies conjugated to biotin are allowed to compete with the unknown analyte and a known quantity of enzyme-labeled analyte. The amount of antibody-analyte complexes is readily determined by insolubilizing the complexes by adding avidin attached to a carrier material. Avidin is bound to a solid support such as particles, filter paper, glass or plastic object by covalent attachment, for example covalent attachment to benzoquinone-activated sepharose.
U.S. Pat. No. 4,582,810 (issued Apr. 15, 1986 to Rosenstein) and PCT Publication 84/03358 (published Aug. 30, 1984) describe the attachment of avidin to latex particles having free carboxyl groups on their surfaces. As described therein, the conventional procedure for covalently attaching avidin to the particles involves the use of a water-soluble carbodiimide in an activation step. While producing reagents, this procedure tends to activate the exposed reactive groups of the protein avidin as well as the carboxyl groups on the particles. The result is intramolecular and intermolecular crosslinking or polymerization of avidin, and a significant portion of the reagent is impaired from complexation with biotin. In addition, there may be premature agglutination of the insolubilized reagent due to the cross-reactivity of the activating compound. These problems present a serious economic loss as well as an impairment of diagnostic sensitivity. It has also been evident that carbodiimides provide a reactive intermediate for avidin attachment which is unstable and must be used immediately.
Various other reagents have been prepared with particles having reactive groups such as epoxides, aldehydes, amino groups and diazonium salts. All of these groups have disadvantages. Epoxide groups are not stable, so that the particles cannot be stored for very long. Particles having aldehyde groups generally tend to agglutinate prematurely. The aldehyde groups also prematurely oxidize, thereby losing binding activity. Particles with amine groups are like the carboxylated materials by requiring an additional activation step for attachment. Diazonium compounds are unstable and therefore undesirable to work with.
Immunological compounds are immobilized on polymer particles having reactive activated 2-substituted ethylsulfonyl and vinylsulfonyl groups to form useful immunological reagents as described in copending U.S. Ser. No. 81,206 filed Aug. 3, 1987 by Sutton and Danielson, now abandoned. In some instances, however, it is not advisable to directly attach immunological compounds to the particles. For example, direct attachment may deactivate the immunological compounds. Yet, it is desired to strongly attach those compounds to the particles.
Hence, reagents which are composed of avidin or biotin covalently attached to a water-insoluble particle would be very useful in diagnostic methods. However, it would be desirable to have such reagents which are readily prepared in an efficient manner and under conditions which are not limiting and which do not reduce sensitivity or generate other undesirable results. It would be particularly desirable to avoid the use of conventional carbodiimide chemistry for attachment whereby premature crosslinking and agglutination are prominent. It would also be desirable to have reagents for insolubilizing immunological species without directly attaching the species to the insoluble carrier material. Yet the resulting attachment should be stronger than that achieved through mere adsorption.